Flavonoids: new ways of thinking in Nutrition

1
Flavonoids new ways of thinking in Nutrition
Cesar G. FragaUniversity of Buenos Aires,
Argentina2004
2
An increase in the consumption of fruit and
vegetables is good for your health. U. S.
National Cancer Institute 5-A-Day Program

• Is equivalent to eat any fruit or vegetable?

If not, is possible to identify the fruits and
vegetables that are healthier?
Is possible to identify and quantify the
substances responsible for such healthy effects?
Are antioxidant substances responsible of such
effects?
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3
French Paradox
In France there is a high intake of saturated
fat but low mortality from CHD.
These paradox can be explained by the high
wine consumption in France.
Renaud S, De Lorgeril M (1992) Lancet 339,
1523-1526
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Phenolic compounds

• Flavonoids ( 4000)

• No flavonoids

More than 4000 flavonoids have been discovered,
and probably exist a lot more
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Flavonoids, flavan-3-ols, and epicatechin
Flavan-3-ol
(-)-Epicatequina
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Catechins structures
(
)
-
C
a
tec
h
i
n

(-
)
-
Ep
i
ca
t
ech
i
n
(-
)
-
Ep
ig
a
l
loca
t
ec
h
i
n
OH
OH
OH
OH
OH
OH
B
O
HO
O
HO
O
HO
OH
C
A
OH
OH
OH
OH
OH
OH


(-
)
-
Ep
i
ca
t
ec
h
in

g
a
l
l
a
te

(-
)
-
Ep
ig
a
l
loc
a
tec
h
i
n

g
a
l
l
a
te
OH
OH
OH
OH
O
HO
O
HO
OH
O
C
O
C
O
O
OH
OH
HO
OH
HO
OH
OH
OH
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Procyanidins
OH
OH
O
HO
OH
OH
OH
n
OH
O
HO
OH
OH
OH
OH
O
HO
OH
OH
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Content of procyanidins in foods and beverages
9
Epidemiología

• Dietary antioxidant flavonoids and the risk of
  coronary heart disease. The Zutphen Elderly
  study. Hertog MGL, et al. (1993) Lancet,
  3421007-1011.
• Flavonoid intake and long term risk of coronary
  heart disease and cancer in the seven countries
  study. Hertog MGL, et al. (1995) Arch Intern Med
  155381-386.
• Dietary flavonoids, antioxidant vitamins, and
  incidence of stroke. The Zutphen study. Keli SO,
  et al. (1996) Arch Intern Med 156637-642.
• Dietary flavonoid intake and risk of
  cardiovascular disease in post-menopausal women.
  Yochum L et al. (1999) Am J Epidemiol 149943-49.
• Dietary catechins and cancer incidence among
  postmenopausal women the Iowa Womens Health
  Study (United States). Arts IC et al. (2002)
  Cancer Causes Control 2002, 13373-382.
• Dietary antioxidants and asthma in adults
  population-based case-control study. Shaheen et
  al. (2001) Am J Respir Crit Care Med
  1641823-1828
• Tea consumption and mortality after acute
  myocardial infarction. Mukamal KJ (2002)
  Circulation 105 2476-2481

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Fisiología

• Endothelin-1 synthesis reduced by red wine.
  Corder R. (2001) Nature, 414863-864
• Selected flavonoids and whole juice from purple
  grapes inhibited platelet and enhance nitric
  oxide release. Freedman JE. et al. (2001)
  Circulation, 1032792-2798
• Short and long term black tea consumption
  reverses endothelial dysfunction in patients with
  coronary artery disease. Duffy SJ, et al. (2001)
  Circulation 104151-156
• Evidence that antioxidant flavonoids in tea and
  chocolate are beneficial for cardiovascular
  health. Kris-Etherton P, Keen CL. (2002) Curr
  Opin Lipidol 1341-49
• Chocolate consumption and platelet function. Holt
  RR et al. (2002) J Am Med Assoc (JAMA)
  2872212-2213
• Flavonoid-rich cocoa induces nitric
  oxide-dependent vasodilation in healthy humans
  Fisher DL et al. (2003) J Hypertension
  212281-2286

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Biochemical mechanisms explaining the antioxidant
effects of flavonoids
1. reaction with free radicals and metal chelation
2. interactions with lipid membranes
3. control of cell redox state
4. regulation of enzyme activity
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1. Reaction with free radicals
There is an extensive literature demonstrating
that flavan-3-ols as well other flavonoids and
polyphenols, has free radical scavenging activity
in vitro.
Studies structure-activity are consistant with a
free radical scavenging action of these
compounds.
Since there is not an absolute measurement of
antioxidant activity, it is very difficult to
establish the potential contribution of a
compound towards the antioxidant defense system
in a non-relative base.
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Reaction of flavan-3-ols with free radicals
Metal chelation by flavan-3-ols, flavonoids, and
polyphenols is supported by their chemical
structures (3, 4 hydroxyl groups).
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Antioxidant activity of procyanidins on lipids,
DNA, proteins, and cells.
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Effect of procyanidins on AAPH-initiated
oxidation in liposomes
AAPH.
40
TBARS ()
20
0
1-mer
2-mer
3-mer
4-mer
5-mer
6-mer
Procyanidin
Lotito et al. (2000) Biochem Biophys Res Commun
276945-951
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Effect of (-)-epicatechin oligomers on DNA
oxidation
Calf thymus DNA was irradiated with UV-C during
15 and 30min in the presence or absence of
different amounts of (-)-epicatechin
Ottaviani y col (2002) Arch Biochem Biophys
406203-208
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Effect of flavanols dimers on albumin thiols
oxidation Albumin was incubated in the presence
or the absence of 5 mM flavanol dimers and 50 mM
AAPH for different times
1
2
0


d
i
m
e
r
s
1
0
0
n
o

a
d
d
i
t
i
o
n


8
0
Remanent SH groups ()
6
0
4
0
2
0
0
0
3
0
6
0
9
0
1
2
0
1
5
0
1
8
0
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Effect of flavanols and procyanidins on cell
oxidation
Jurkat cells were incubated for 24 h in the
presence of different amounts of flavanol dimers,
and then during 60 min in the presence of 1 mM
AMVN at 37ºC
25 mg/ml
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Actis-Goretta et al., Res Commun Pharmacol
Pathol, in pess
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Catechins can interact with other antioxidants,
and they do it according to their physical and
chemical properties (reduction potentials,
solubility, hydrophobicity, etc.)
Ascorbic acid, a-tocopherol, and epicatechin
consumption and TBARS formation.
Lotito y Fraga (1998) Free Radic Biol Med
24435-441 Lotito y Fraga (2000) Proc Soc Exp
Biol Med 22532-38
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2. Interaction with lipid membranes
Oxidants, Triton X-100)
Membrane damage
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(-)Epicatechin, ()catechin, and the procyanidins
did not affect membrane physical properties that
could modulate lipid oxidation rates (fluidity,
lateral phase separation).
Verstraeten y col (2003) Free Radic Biol Med
3484-92
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Evaluation of liposome-micelle transition Effect
of the size of the molecule
RF
1.0
300
RF
0.5
200
EC50 ( change)

100
0
0
1
2
3
4
5
6
Monomer units
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Triton X-100 (mM)
Verstraeten y col (2003) Free Radic Biol Med
3484-92
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3. Control of cell redox state
Concentration of ()-catechin, (-)-epicatechin
and dimer in the nuclei of Jurkat cells
p107 Jurkat cells were suspended in 10 ml of media
supplemented with ()-catechin, (-)-epicatechin,
or dimers, and incubated for 24h at 37C.
Catechin, epicatechin and dimer were determined
by HPLC.
Mackenzie et al (2004) FASEB J 17 1096-1098
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NF-kB-DNA binding activity in nuclear fractions
of Jurkat cells
36
24

(Arbitrary units)
Binding acivity

12
NF-kB
0
C
PMA
1.7
4.3
8.6
17
Dimers (mM)
Cells were incubated in the absence (C, PMA) or
the presence of 1.7-17 µM Dim for 24 h, followed
by 4 h of incubation without or with PMA. DNA
binding activity of NF-kB was determined by EMSA
assay (n 6).
Mackenzie et al (2004) FASEB J 17 1096-1098
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Dimers, (-)-epicatechin, and ()-catechin inhibit
IL-2 release from Jurkat cells
Cells were incubated in the absence (C, PMA) or
the presence of 17 µM Dim, 8.6 µM Epi, or 17 µM
Cat for 24 h, followed by 18 h of incubation
without or with PMA. IL-2 was determined by ELISA
(n 4).
Mackenzie et al (2004) FASEB J 17 1096-1098
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4. Regulation of enzyme activity
Enzyme activities inhibited by flavonoids

• Glutathione reductase
• Epoxide hydrolase
• Glioxalase
• Xanthine oxidase
• Aromatase
• 11-b-hydroxysteroid dehydrogenase
• Catechol-o-methyltransferase
• Aldose reductase
• Monoamine oxidase (FAD)
• Aldo-keto reductase )(family)
• Hyalouronidase
• RNA/DNA polymearse
• Lactate dehydrogenase
• Piruvate dehydrogenase
• Aldehyde/alcohol dehydrogenase
• Elastase
• Sialidase

• Kinases
• Phospholipase A2
• ATPases
• Lipoxygenases and cyclooxygenases
• Phospholipase C
• Cyclic nucleotide phosphodiesterase
• Adenylate cyclase
• Reverse transcriptase
• HIV-1 proteinase
• HIV-1 integrase
• Ornithine decarboxylase
• Topoisomerase
• Histidine (DOPA) decarboxilase
• Malate dehydrogenase
• Cyt P450 systems
• Nitric oxide synthase
• Cyclooxygenase

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Effect of flavan-3-ols and procyanidins on ACE
activity in rat kidney homogentes
Flavan-3-ols and procyanidins were assayed at 100
µM (substrate concentration 5 mM) .
Actis Goretta et al. 2003, FEBS Lett 555, 597-600
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Kinetic parameters for the ACE inhibition by
(-)-epicatechin, dimers and hexamers fractions
Actis Goretta et al. 2003, FEBS Lett 555, 597-600
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Ancillary bibliograghy

• Flavonoids antioxidants or signalling molecules?
  Williams RJ et al. (2004) Free Rad Biol Med 36
  838-849
• Metabolism of dietary procyanidins in rats.
  Gonthier M-P et al. (2003) Free Rad Biol Med 35
  837-844
• Structure-activity relationshipsgoverning
  antioxidant capacities of plant polyphenols. Bors
  W et al. (2001) Meth Enzymol 335166-180
• The effects of plant flavonoids of mammalian
  cells implications for inflammation, heart
  disease, and cancer. Middlenton Jr E (2000)
  Pharmacol Rev 52673-751
• Flavonoids a review of probable mechanisms of
  action and potential applications. Nijveldt RJ
  (2001) Am J Clin Nutr 74418-425

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Flavonoids, nutrients and antioxidants
(Conclusions)
1. Flavonoids can act as antioxidants in vivo.
2. Beyond the flavonoid capacity to scavenge free
radicals and/or chelate metals, other mechanisms
can explain the antioxidant activity of
flavonoids.
3. Flavonoids absorption and bioavailability are
a major consideration when assessing the health
and antioxidant effects of flavonoids.
Fraga-2004